KR101998246B1 - A pharmaceutical composition for treating cancer comprising an ionic compound having metal ions binding thereto - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating cancer. More specifically, the pharmaceutical composition for treating cancer contains an ionic compound in which two compounds selected from ascorbic acid, dichloroacetic acid, and lactate are combined with calcium ions. Since compounds different from each other are uptaken into cancer cells simultaneously, compounds act on major metabolic enzymes with different mechanisms of cancer cells simultaneously to duplicately and complexly disturb a metabolic process of cancer cells, thereby having more excellent treatment effect compared to an anticancer agent focusing on one specific mutation or cancer cell growth signal. In addition, the pharmaceutical composition is less susceptible to drug resistance, thereby more effectively inhibiting the action of cancer cells, such as proliferation, invasion, metastasis and the like.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for treating cancer comprising an ionic compound bonded to a metal ion,

The present invention relates to a pharmaceutical composition for treating cancer, and more specifically, a pharmaceutical composition for treating cancer comprises an ionic compound in which two compounds selected from ascorbic acid, dichloroacetic acid and lactate are combined with one metal ion , The different compounds are simultaneously uptaked into cancer cells, which act through the different mechanisms in cancer cells to duplicate and complex the metabolic processes of cancer cells, resulting in one particular mutation or cancer cell growth signal The present invention relates to a pharmaceutical composition for treating cancer which is more effective than an anticancer agent of the present invention and is less susceptible to the occurrence of drug resistance, thereby more effectively inhibiting the action of proliferation, invasion and metastasis of cancer cells.

In general, there are three methods of treating cancer: surgical surgery, radiation therapy, and chemotherapy. Each method can be used independently for cancer treatment or a combination of two or more methods. Many early stage cancers can be treated with surgical surgery, but if the cancer has progressed much or if metastasis has occurred, surgical treatment alone will be difficult and should be done with other methods. Radiation therapy is used for the treatment of cancer that is particularly reactive to areas of difficult surgical operation or radiation, and may be used in conjunction with medication or before or after surgery. Radiation therapy, however, has the disadvantage of causing damage to the normal skin of the local area due to the high energy side effects of radiation, and metastatic cancer has the disadvantage that cancer stem cells are resistant to radiation and subsequently recurred or metastasized. To treat cancer with high mortality, surgical treatment, chemotherapy or radiotherapy are the top priority in early and middle stage cancer. However, current cancer therapies are generally only capable of treating early cancers, have a high possibility of recurrence, and have various side effects such as destroying normal cells in addition to cancer cells. Especially in patients with severe end-stage cancer, the side effects of aggressive therapies may be relatively more serious, so that treatment methods that slow the progression of cancer cells and reduce side effects and improve quality of life are often chosen.

In general, chemotherapy is a method of destroying or inhibiting DNA or related enzymes necessary for the proliferation of cancer cells by injecting the drug with a light or injection. Chemotherapy is being used as a standard therapy for metastatic cancer treatment in that the cancer can be reached in any part of the body compared to radiation therapy or surgery, and the drug can reach and treat the metastatic cancer. Of course, it is not possible to cure metastatic cancer by chemotherapy, but it plays an important role in alleviating symptoms and improving quality of life and prolonging the life span of the patient. However, the problem with most chemotherapeutic agents is not only cancer cells but also normal cells, especially bone marrow, hair follicles, and gastrointestinal endothelial cells, which are proliferating in the human body. Therefore, Such as bacterial infection, natural hemorrhage, hair loss, nausea and vomiting, due to the decrease of white blood cells and platelets. Also, due to the manifestation of drug resistance, the effect initially appears to fail and eventually fails. Immunologic chemotherapy, which is a tailor-made treatment to treat cancer by enhancing immunity using gene technology, is not easy to remove cancer cells because of its immune function, Patients, PD-1 (proteins present on the surface of activated T cells) are disadvantaged to patients who do not express much, and large-scale production is not possible and significant cost is required. There is a possibility.

Recently, there has been an increase in the number of targeted anticancer drugs that selectively kill only cancer cells while reducing the adverse effects of existing anticancer drugs and protect normal cells. However, since only specific factors of cancer development process are attacked, even cancer of the same kind is effective only for patients showing specific target factors . Although many anticancer drugs based on continuous cell proliferation and metastasis inhibition, which are characteristic of cancer cells, have been developed for treating cancer for a long time, they have developed an anticancer drug that effectively inhibits the growth of cancer cells controlled by complex network of signal transduction pathways Is still a very difficult task.

It is urgent to develop new therapies that are easily applicable to the treatment of cancerous diseases and can be effectively treated while minimally affecting normal tissues.

In order to satisfy this demand, novel anticancer agents using the inherent metabolic characteristics of cancer cells have been attracting attention. With the development of genetic engineering and molecular biology techniques in the 1970s, cancer mutations and chromosomal abnormalities were discovered, and the focus of cancer research was focused on the genetic causes of cancer, and the unique metabolic pathway of cancer is not the cause of cancer, And it has not been studied for a long time. However, it has been shown that genes related to cancer metabolic signals and various metabolites can directly induce cancer. With the development of biotechnology, it is possible to analyze the metabolites, so that the metabolic pathway of cancer cells can treat cancer With a strong anti-cancer target. The first to discover that cancer cells use a different metabolic pathway than normal cells suggests that cancer cells use a metabolic pathway through a new pathway - the Aerobic glycolysis (Warburg effect) - and the Nobel Prize winning German biochemist It was by Otto Warburg.

Normal cells produce energy by completely oxidizing glucose to water and carbon dioxide by oxidative phosphorylation in the presence of oxygen, while cancer cells oxidize glucose to pyruvate in an oxygen-deficient environment (Hypoxia) And then select a pathway for reduction to lactate (lactate). Therefore, cancer cells have less oxygen consumption than normal cells, and a large amount of lactate exists in the ascites fluid of cancer patients. Cancer cells consume a greater amount of glucose than normal cells, (ATP) through a glycolytic process that produces a large amount of ATP.

In many cases, cancer cells, particularly solid cancer cells, use the action as a metabolic pathway for production of energy source (ATP), including mitochondrial defects and dysfunction, adaptation to the hypoxic microenvironment of the tumor, And abnormal expression of metabolic enzymes. The Warburg effect is the result of adaptation of cancer cells to the hypoxia microenvironment. The hypoxic environment inhibits the ubiquitous proteolytic degradation of HIF1 (a transcription factor that is induced when cells are deficient in oxygen) Stabilizes HIF1 and induces activation as a transcription factor. On the other hand, GLUT1 (protein type 1 transporting sugar) is induced by HIF1, The MCT4 (monocarboxylic acid transporter) also supports the entry of glucose into cancer cells, and lactate dehydrogenase A (LDHA), which converts pyruvic acid to lactate as a direct target of HIF1, To the outside of the cancer cell. In addition, HIF1 induces the expression of Pyruvate Dehydrogenase Kinase (Pyruvate Dehydrogenase Kinase), which inhibits pyruvate dehydrogenase (PDH), an enzyme that converts pyruvic acid to Acetyl-Co to shut down the pathway to oxidative phosphorylation . Thus, HIF1 is a very important factor in inducing the Warburg effect through regulation of glucose uptake and direct expression of various factors related to the process.

On the other hand, cancer cells rapidly excrete lactate, which is the final product of the action, to prevent acidification by themselves. Lactate released is produced in cytotoxic T cells and dendritic cells, And inhibits the expression of NKp46 (a cognitive receptor of natural killer cell, NK cell), which is an activating receptor of natural killer cells, and inhibits the production of immunosuppressive and inhibitory substances To inhibit the apoptosis of cancer cells. In addition, the endothelial cells surrounding the cancer cells contain IL-8 (a protein that acts as a chemoattractant factor that activates inflammatory cells and induces them to inflammation sites) and VEGF (an angiogenic factor) Induces the expression of endothelial cells and promotes the migration of vascular endothelial cells, resulting in induction of angiogenesis. Metabolic reprogramming of these cancer cells is an evolutionarily selected metabolic transformation strategy to produce precursors such as nucleotides, lipids, amino acids, etc. necessary for the synthesis of cellular components of rapidly growing cancer cells rather than simply producing ATP, It is understood that growing cancer cells strategically utilize this metabolic pathway. Thus, the development of cancer by various cancer-causing factors that induce the formation and growth of cancer is closely related to the cancer cell metabolism, and reprogramming of the cell metabolism may be an important cancer target to effectively treat cancer . Understanding the specific metabolic signals of cancer cells alone and selectively eliminating the tumors, the development of metabolic target therapeutic agents for controlling the glucose metabolism of cancer cells is currently being intensively carried out. Especially, the various metabolites used for glucose metabolism and infectious diseases Development of a cancer treatment agent that effectively uses drugs is being carried out.

Korean Patent Laid-Open Publication No. 10-2016-0082918 (July 28, 2016) U.S. Patent Application Publication No. US2011 / 0117210 (May. 19, 2011) Korean Patent Publication No. 10-2005-0058278 (2005. 06. 16.)

The inventors of the present invention conducted various studies to develop a cancer-targeting therapeutic agent and a therapeutic method capable of selectively removing the tumor based on the understanding of specific metabolic signals of cancer cells only. As a result, ascorbic acid, dichloroacetic acid and lactate It is confirmed that the use of an ionic compound in which two kinds of compounds selected from the group consisting of Ca, Zn, Mg, and Fe are combined with one metal ion can effectively inhibit the proliferation, invasion and metastasis of cancer cells Thereby completing the invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for treating cancer, which comprises an ionic compound in which two selected compounds of ascorbic acid, dichloroacetic acid and lactate are combined with one metal ion selected from Ca, Zn, Mg and Fe To provide a composition.

In order to achieve the above object, the present inventors paid attention to a major metabolic pathway peculiar to cancer cells in order to develop a method for effectively inhibiting cancer cell proliferation and metastasis.

First, the metabolic pathway of cancer cells that are noticed is aerobic glycolysis, and cancer cells mainly use the action that does not require oxygen rather than the oxidative phosphorylation, which is an energy metabolism process that requires oxygen, Cells are able to survive even in a hypoxia environment such as immature solid cancers and inactivate the process of cell death control starting from mitochondria.

Secondly, the metabolic pathway of cancer cells is related to a large amount of lactate produced through the action. Lactate is rapidly released from the cancer cells to prevent the cancer cells themselves from being acidified, It is made into acidosis and the activity of NK and CTL cells is inhibited by the acidified surrounding environment and eventually induces an angiogenesis, cancer cell metastasis and immunosuppression.

Third, calcium plays an important role in the cytosolic calcium buffering, which is essential for the survival and proliferation of cancer cells, and plays an important role in cell apoptosis and autophagy associated with active oxygen production, It is also important to note that nutritional value is high. In particular, calcium is known to be maintained at low concentrations in cancer cells. Reducing the supply of calcium to mitochondria in cancer cells inhibits cancer cell proliferation by energy depletion. Conversely, increasing calcium supply leads to overloading of mitochondria resulting in the death of cancer cells. Therefore, it was noted that cancer cells respond more sensitively to calcium than normal cells, so that the destruction of calcium homeostasis in cancer cells kills the growth of cancer cells beyond the suppression of cancer cells.

The present invention is based on the finding that two compounds selected from among ascorbic acid, dichloroacetic acid and lactate, which are ionic compounds which act effectively on the major metabolic pathway peculiar to cancer cells, are one kind of metal ion selected from Ca, Zn, The present invention provides a pharmaceutical composition for treating cancer comprising an ionic compound combined with an ionic compound.

The pharmaceutical composition for cancer treatment according to the present invention is characterized in that an ionic compound in which two kinds of compounds selected from ascorbic acid, dichloroacetic acid and lactate are combined with one metal ion selected from Ca, Zn, Mg and Fe as an active ingredient And can effectively inhibit the proliferation of cancer cells.

In addition, the pharmaceutical composition for cancer treatment according to the present invention includes compounds having different mechanisms such as a cancer cell growth inhibiting compound and a cancer cell transfer inhibiting compound, thereby simultaneously acting on major metabolic enzymes to duplicate the metabolic processes of cancer cells Unlike conventional anticancer drugs, which are focused on one specific mutation or blockade of metabolic pathway, the drugs can be used simultaneously at the cellular level.

In addition, the pharmaceutical composition for treating cancer according to the present invention is an ionic compound, which can improve the uptake of cancer cells when administered into the body.

In addition, the pharmaceutical composition for cancer treatment according to the present invention can improve the uptake of cancer cells by converting an acid compound into a neutral metal salt form, and is less susceptible to the occurrence of drug resistance and exhibits effects such as proliferation, invasion, Can be effectively suppressed.

In addition, the method for treating cancer and the method for inhibiting cancer metastasis using the pharmaceutical composition for treating cancer according to the present invention can be provided.

In addition, the composition for inhibiting cancer metastasis or improving cancer, including the pharmaceutical composition for treating cancer according to the present invention, can be provided.

In addition, the pharmaceutical composition for treating cancer according to the present invention has low side effects and can be used as a food additive and can be administered at a high dose.

FIG. 1A is a graph showing calcium concentrations in cancer cells treated with calcium salts of Examples 1 to 3. FIG.
Fig. 1B is an image showing calcium in cancer cells treated with the calcium salts of Examples 1 to 3. Fig.
FIG. 2 is a graph showing lactate concentrations in cancer cells treated with calcium salts of Examples 1 to 3. FIG.
FIG. 3 is a graph showing ascorbic acid concentrations in cancer cells treated with calcium salts of Examples 1 and 2. FIG.
FIG. 4 is a graph showing pH in cancer cells treated with calcium salts of Examples 1 to 3. FIG.
FIG. 5 is a graph showing the concentrations of pyruvic acid in cancer cells treated with calcium salts of Examples 1 to 3. FIG.
6 is a graph showing the concentration of? -GK in cancer cells treated with calcium salts of Examples 1 to 3;
FIG. 7 is a graph showing the expression levels of PARP, β-catenin, VEGF and β-actin expressed from cancer cells treated with calcium salts of Examples 1 to 3.
8 is a graph showing the expression levels of reactive oxygen species expressed from cancer cells treated with the calcium salts of Examples 1 to 3. FIG.
FIG. 9 is a graph showing the self-destruction of cancer cells treated with calcium salts of Examples 1 to 3. FIG.
10 is an image obtained by treating the calcium salts of Examples 1 to 3 with a colon cancer cell line to confirm the cell cluster formation ability
FIG. 11 is a graph showing the cell populating ability by administering the calcium salt of Examples 1 to 3 and the conventional 5-FU anticancer agent in combination with a colon cancer cell line.
12A is a photograph of the imaging result observed after incising the calcium salt of Example 1 in a mouse model (DLD-1 orthotopic model) after 1 week, FIG. 12B is a photograph of the calcium salt of Example 1 in a mouse model (DLD-1 orthotopic model) and the weight of cancer tissue cut after 1 week.
FIG. 13 is a photograph of the growth saturation of cancer obtained by luminescence imaging measurement after administration of calcium salts of Examples 1 to 3 to a mouse model transplanted with A549 / LUC cells into lungs.
FIG. 14 is a graph showing the ROI (Region Of Interest) of the program of the IVIS spectrum (Xenogen) measured by the image of FIG.
FIG. 15 is a graph showing the survival rate after administering the calcium salts of Examples 1 to 3 to a mouse model transplanted with A549 / LUC cells in the lungs. FIG.

The present invention relates to an ionic compound in which two kinds of compounds selected from among ascorbic acid, dichloroacetic acid and lactate are combined with one kind of metal ion selected from Ca, Zn, Mg and Fe As an active ingredient.

L-ascorbic acid is a vitamin C that has already been identified as a non-toxic anticancer drug through the study of Nobel Prize winner Linus Pauling. In addition, ascorbic acid does not show any toxicity when administered to the human body in an excess amount (over 50 g), and it can competitively inhibit the glucose and glucose addition of cancer cells due to a structure similar to that of glucose. On the other hand, ascorbic acid at a high concentration (mega dose) can induce necrosis of cancer cells by decreasing glutathione and NADPH in cancer cells and generating reactive oxygen species (ROS). In addition, ascorbic acid can induce cancer cells to differentiate into normal cells, inhibiting the spread of cancer cells to cancer tissues through blocking of enzymes that help collagen synthesis and cancer metastasis. In addition, it enters the cancer cell, destroys the cell membrane, reduces the cancer pain, and detoxifies important organs of the cancer patient to improve immunity and inhibit cancer angiogenesis. It can also play an important role in cancer prevention and treatment by increasing the efficacy of other anticancer drugs or radiation therapy and by reducing side effects.

On the other hand, when the ascorbic acid was low in concentration, no auto-apoptosis of cancer cells was observed but the complete growth was inhibited at the G1 stage of cancer cells, p53 level was increased, CDK2 activity was inhibited, and p38MARK activation and COX-2 expression .

When the ascorbic acid concentration is high, the decrease of the dislocation of the micondriac membrane and the expression of the Tf transport receptor decrease, and the induction of apoptosis of cancer cells through reduction of iron absorption and increase of reactive oxygen species (ROS) .

When the ascorbic acid is combined with an appropriate metal ion, the stability in the body is enhanced and the uptake into the cancer cell is increased, so that it is more improved than the anticancer effect of the existing ascorbic acid and can induce the self-death of the cancer cell at a relatively low concentration .

Generally, when cancer cells become hypoxic, hypoxia-inducible factor-1 (HIF-1) is activated and pyruvate dehydogenase kinase is expressed and expressed by pyruvate kinase The pyruvate dehydrogenase complex is inhibited. As a result, pyruvic acid is not converted to acetyl-CoA, pyruvic acid is accumulated, and mitochondrial energy synthesis is degraded. When pyruvate is accumulated in excess, pyruvate is converted to lactate, and lactate accumulates around cancer cells. Taken together, in short, when cancer cells become hypoxic, accumulation of lactate begins, starting with the expression of pyruvate kinase.

The dichloroacetic acid is non-toxic and can block the aerobic glycolysis pathway described above. In addition, the inhibition of the expression of pyruvate kinase can inhibit accumulation of lactate. In addition, the TCA circuit (Tricarboxylic Acid Cycle) can be regained to induce glucose metabolism reprogramming (ie normalization of mitochondrial metabolism) by the respiration enhancement of the mitochondria.

On the other hand, the dichloroacetic acid can bind to an appropriate metal ion to enhance the anticancer effect of conventional dichloroacetic acid, induce normalization of mitochondrial metabolism, and induce reactive oxygen species (ROS) to kill cancer cells.

In addition, the dichloroacetic acid can inhibit acidosis of cancer cells by reducing the accumulation of lactate by binding with an appropriate metal ion.

The lactate includes lactic acid, D-lactate and L-lactate, and also includes D-lactic acid and L-lactic acid.

By linking the lactate with an appropriate metal ion, lactate in cancer cells is accumulated excessively to activate LDHB (L-lactate dehydrogenase B, an enzyme that converts lactate or lactate into pyruvic acid and simultaneously converts NAD + into NADH) LDH (L-lactate dehydrogenase A; LDHB reverse-reaction enzyme) is inhibited and can inhibit the expression of MCT (monocarboxylate transporters).

Here, "suppression of LDHA" or "activation of LDHB" means that lactate is converted to pyruvic acid. In addition, "suppression of expression of MCT" means that the expression of NKp46 is activated by inhibiting the expression of MCT involved in lactate inflow and excretion, thereby activating the cell apoptosis of cancer cells.

Further, the lactate can be introduced into cancer cells by binding with suitable metal ions, and the inside can be acidified to induce apoptosis.

The metal ion may be one selected from Ca, Zn, Mg, and Fe. But is preferably Ca, Mg, Fe, more preferably Ca ²⁺ ion, but is not limited thereto.

In this case, the Ca ^{2 +} ion (calcium ion) is to affect the calcium in the cancer cell homeostasis (Homeostasis), to induce a calcium accumulation in the mitochondrial cancer can generate an excess of free radicals inside the cell, the generated active oxygen Lt; RTI ID = 0.0 > cell death. ≪ / RTI >

More specifically, cancer cells are involved in energy production. In mitochondria, calcium is directly involved in alpha-ketoglutarate dehydrogenase and is an important factor in the normal operation of TCA circuits. Is particularly important in reducing the number of patients. Excessive increase of calcium concentration in cancer cells activate endonuclease and many proteases, causing metabolic disturbances of mitochondria, while cytochrome C is advantageous and caspase 9 is activated to activate caspases 3 and 7. This leads to apoptosis.

In the present specification, the term "ionic compound " refers to a compound in which ions having charges opposite to each other by electrostatic force are formed through ionic bonding, and the compound generally exhibits electrical neutrality.

The ionic compounds included in the pharmaceutical compositions according to the present invention are preferably selected from the group consisting of calcium salts of ascorbic acid and dichloroacetic acid, calcium salts of ascorbic acid and lactate, calcium salts of dichloroacetic acid and lactate, Magnesium salts of coric acid and dichloroacetic acid, magnesium salts of ascorbic acid and lactate, magnesium salts of dichloroacetic acid and lactate, magnesium salts of ascorbic acid and dichloroacetic acid, magnesium salts of ascorbic acid and lactate , Magnesium salts of dichloroacetic acid and lactate, iron salts of ascorbic acid and dichloroacetic acid, iron salts of ascorbic acid and lactate, iron salts of dichloroacetic acid and lactate, more preferably Include calcium salts of ascorbic acid and dichloroacetic acid, ascorbic acid and lactate Calcium salt of lactose, dichloroacetic acid and calcium salt of lactate. But is not limited thereto.

The term " calcium salt "as used herein means an ionic compound produced or synthesized in a form in which a compound is bonded to calcium ions, and the" magnesium salt "means an ionic compound produced or synthesized in a form in which a compound is combined with magnesium ion Refers to an ionic compound produced or synthesized in the form in which a compound is bonded to an iron ion.

The pharmaceutical composition according to the present invention can be used for radiation therapy or in combination therapy with an anticancer agent. In general, since the expression of PARP, HIF-1α and VEGF, which confer radiation resistance to cancer cells upon irradiation, is reduced, the combined use of radiation and irradiation enhances the anticancer activity of radiation, , While exhibiting an equivalent level of anticancer activity. In this case, the dose of radiation which can be used is not particularly limited, but may be 2 to 10 Gy per day, and the radiation may be irradiated once a day or divided into several days.

The pharmaceutical composition according to the present invention comprises an ionic compound in which two selected compounds of ascorbic acid, dichloroacetic acid and lactate are combined with one metal ion selected from Ca, Zn, Mg and Fe, The compounds can be simultaneously uptaked in cancer cells without being counteracted by the respective anticancer effects of the compounds. This effect can be more effective than the conventional combination therapy (Combi-therapy).

On the other hand, when the pharmaceutical composition according to the present invention and the anticancer agent are administered in combination, the anticancer agent can exert more excellent anticancer effect than the anticancer agent administered with the single anticancer agent.

At this time. The anticancer agent that can be used in combination with the pharmaceutical composition according to the present invention is not particularly limited as long as it does not directly act on the whole metabolic process of cancer cells. For example, imatinib, 5-FU (5- Such as erythromycin, Florouracil, Irinotecan, Sunitinib, Oxaliplatin, Paclitaxel, Lapatinib, Trastuzumab, Herceptin, Gefitinib, Erlotinib ), Methotrexate, Carboplatin, Docetaxel, Everolimus, Sorafenib, inhibitors of carbonic anhydrase, monocarboxylate trans Pabrolizumab, Atezolizumab, PD-1 anticancer drug, Nivolumab, PARP-1 (poly (ADP-ribose) polymerase 1) inhibitors of monocarboxylate transporter Inhibitor, PARP-2 (P Olaparib, Rucaparib, Niraparib, Bevacizumab and VEGF inhibitors as well as other anticancer agents known to exhibit anticancer activity, as well as inhibitors of the ADP-ribose polymerase 2 .

In the present invention, the cancer is a cancer that can be inhibited by proliferation, invasion, metastasis, etc. due to disturbance of the metabolic process. Examples of the cancer include lung cancer, breast cancer, colon cancer, gastric cancer, brain cancer, pancreatic cancer, thyroid cancer, Uterine cancer, cervical cancer, renal cancer, and melanoma.

The pharmaceutical composition of the present invention may be prepared in the form of a pharmaceutical composition for treating cancer, which further comprises an appropriate carrier, excipient or diluent conventionally used in the production of the pharmaceutical composition. Specifically, the pharmaceutical composition may be formulated into oral compositions, external preparations, external patches, suppositories and sterilized injection solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, And the like.

In the present invention, the carrier, excipient and diluent which may be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include tablets, depots, pills, powders, granules, capsules, oral patches and the like, which may contain at least one excipient in the extract and its fractions, for example, Starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have. Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, external patches, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The content of the ionic compound contained in the pharmaceutical composition of the present invention is not particularly limited but may be in the range of 0.0001 to 50% by weight, more preferably 0.01 to 20% by weight, based on the total weight of the final composition, The concentration of the metal ion in a single dose of the pharmaceutical composition may be from 0.1 to 300 mM.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " of the present invention means a therapeutic or prophylactic treatment of a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention And the effective dose level refers to the level of the disease to be treated, the severity of the disease, the activity of the drug, the age, body weight, health, sex, sensitivity of the patient to the drug, Duration, duration of administration, factors involved in combination with or contemporaneously with the composition of the present invention, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered alone or in combination with a known anticancer agent or an ingredient known to exhibit anticancer activity. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.

The dosage of the pharmaceutical composition of the present invention can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction to the disease, the age, body weight, sex, history, or kind of the substance used as the active ingredient. For example, the pharmaceutical composition of the present invention may be administered at about 1 ng to about 2,000 mg / kg, preferably 1 mg to about 400 mg / kg per adult, and the frequency of administration of the composition of the present invention is particularly It is not limited, but it can be administered once a day or divided into several doses. The dose or the number of administration does not limit the scope of the present invention in any aspect.

In another aspect, the present invention provides a method for treating cancer, comprising administering the pharmaceutical composition to a subject suffering from cancer in a pharmaceutically effective amount.

The term "individual" of the present invention may include, without limitation, mammals, including fish, including rats, livestock,

The term "treatment" of the present invention means any action that improves or alleviates the symptoms of cancer by administering a pharmaceutical composition containing the ionic compound of the present invention as an active ingredient to an individual suffering from cancer.

In the method of treating cancer of the present invention, the kind of cancer to be treated is the same as that described above.

The composition may be administered in single or multiple doses in a pharmaceutically effective amount. At this time, the composition may be formulated and administered in the form of a liquid preparation, a powder, an aerosol, an injection, a solution (a ring gel), a capsule, a pill, a tablet, a suppository or a patch.

The administration route of the pharmaceutical composition for cancer treatment of the present invention can be administered through any ordinary route as long as it can reach the target tissue.

The pharmaceutical composition of the present invention may be administered orally, intraperitoneally, intramuscularly, subcutaneously, intradermally, percutaneously, orally, intranasally, intracorporally, rectally ≪ / RTI > and the like. However, since the lactate metal salt can be denatured or degraded by gastric acid, it can be administered in the form of an unformulated form when it is orally administered. Therefore, the oral composition can be formulated so as to be coated with an active agent or protected from decomposition Or in the form of an oral patch. In addition, it can be administered in a long acting injection mode for maximizing the efficacy upon injection administration. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

In addition, the pharmaceutical composition of the present invention can be formulated into a sustained-release preparation, thereby effectively maintaining the concentration of the drug in the body, that is, the ionic compound. For example, once a day or once a week, the rate of release of the drug in the body can be controlled while maintaining the drug efficacy. At this time, the sustained-release preparation may contain a carrier, an excipient and a diluent as described above.

In another embodiment of the present invention, cancer metastasis comprising an ionic compound wherein two selected compounds of ascorbic acid, dichloroacetic acid and lactate are combined with one metal ion selected from Ca, Zn, Mg, and Fe, Or a pharmaceutically acceptable salt thereof.

The ionic compound provided in the present invention can inhibit various characteristics that can induce cancer cell metastasis such as metastasis, invasion, angiogenesis, and colonization ability of cancer cells, Can be used.

The ionic compound and the metal ion are the same as described above.

The pharmaceutical composition for inhibiting metastasis may be one selected from the group consisting of metastatic lung cancer, breast cancer, colon cancer, stomach cancer, brain cancer, pancreatic cancer, thyroid cancer, skin cancer, bone cancer, lymphoma, And melanoma. ≪ Desc / Clms Page number 2 >

In another embodiment, the present invention provides a method for inhibiting metastasis of cancer comprising administering the pharmaceutical composition to a subject in which a metastasis of cancer is expected to be in a pharmaceutically effective amount.

The term "metastasis" of the present invention refers to the condition of spreading to other tissues away from organs of cancer or malignancy.

When the ionic compound provided in the present invention is administered, the above transition can be inhibited.

In the method for inhibiting cancer metastasis of the present invention, the kind of cancer to be transfected, the type of drug to be administered, the route of administration of the drug, and the like are the same as those described above.

Another aspect of the present invention provides a pharmaceutical composition for preventing or ameliorating cancer-related fatigue comprising the ionic compound as an active ingredient.

Here, cancer-related fatigue is one of the most frequently occurring side effects during or after treatment of cancer, and cancer-related fatigue (CRF) is one example. Here, cancer fatigue syndrome refers to symptoms that are painful, persistent, irrelevant to recent activities, and interfere with daily function, due to the subjective sensation of fatigue and discomfort associated with cancer and its treatment.

In general, when cancer patients become inadequate as ascorbic acid, the permeability of the brain-blood barrier increases, causing neurotoxic substances or viruses to easily invade the brain, thereby causing various fatigue syndromes. In addition, when ascorbic acid becomes insufficient, Neurotoxic substances such as drenochrome are increased, resulting in organ damage.

Accordingly, the ionic compound according to the present invention is prepared by binding a metal ion to two or more compounds containing ascorbic acid. When applied to a cancer patient, it acts to restore immune function, reduce muscle pain, And the cancer fatigue syndrome can be prevented or improved. This effect can also increase the survival rate of cancer patients.

According to another embodiment of the present invention, there is provided a food composition for cancer improvement comprising the ionic compound as an active ingredient.

At this time, the ionic compound is the same as described above.

The ionic compound can be prepared and taken in the form of a food which can be remodeled and which can improve cancer. At this time, the content of the calcium salt contained in the food is not particularly limited, but may be, for example, 0.001 to 10% by weight, and as another example, 0.1 to 1% by weight based on the total weight of the food composition. When the food is a beverage, it may be contained in an amount of 1 to 10 g based on 100 ml as an example, and 2 to 20 g as another example.

In addition, the composition may contain additional ingredients which are commonly used in food compositions and which can improve odor, taste, vision and the like. For example, vitamins A, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid and the like. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn) and copper (Cu) It may also include amino acids such as lysine, tryptophan, cysteine, valine, and the like. In addition, it is also possible to use antiseptics (such as potassium sorbate, sodium benzoate, salicylic acid, and sodium dehydroacetate), disinfectants (such as bleaching powder and highly bleached white powder, sodium hypochlorite), antioxidants (butylhydroxyanilide (BHA), butylhydroxytoluene BHT), etc.), coloring agents (such as tar pigments), coloring agents (such as sodium nitrite and sodium acetic acid), bleaching agents (sodium sulfite), seasoning (such as MSG sodium glutamate), sweeteners (such as hypoglycemia, , Food additives such as flavorings (vanillin, lactones, etc.), swelling agents (alum, potassium hydrogen D-tartrate), emulsifiers, emulsifiers, thickeners, encapsulating agents, gum bases, foam inhibitors, ) Can be added. The additives are selected according to the type of food and used in an appropriate amount.

On the other hand, a functional food for cancer improvement can be prepared using the ionic compound as a food composition for cancer.

As a specific example, the food composition can be used to produce processed foods that can improve cancer, such as confectionery, beverages, liquors, fermented foods, canned foods, processed milk products, processed meats or processed noodles ≪ / RTI > health functional food. The sweets include biscuits, pies, cakes, breads, candies, jellies, gums, cereals (including dinner utensils such as cereal flakes). Drinks include drinking water, carbonated beverages, functional ionic beverages, juices (such as apples, pears, grapes, aloes, citrus fruits, peaches, carrots, tomato juices, etc.) and sikhye. The mainstream includes sake, whiskey, shochu, beer, liquor, and fruit wine. Fermented foods include soy sauce, miso, and kochujang. Canned products include canned goods (for example, tuna, mackerel, saury, canned fish, etc.), canned products (beef, pork, chicken and turkey canned products) and agricultural products (canned products such as corn, peach and canned pineapple). Milk processed foods include cheese, butter, yogurt and the like. Meat-processed foods include pork cutlet, beef cutlet, chicken cutlet, sausage. Sweet and sour pork, nuggets, and nubani. Noodle processed foods include dried noodles, somen noodles, ramen noodles, udon noodles, cold noodles, and sealed noodles. In addition, the composition may be used in retort food, soup and the like.

The term "functional food " of the present invention refers to a food for special health use (FoSHU) Refers to a food having a high content. The food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, rings and the like in order to obtain a useful effect for improving cancer.

EXAMPLES Hereinafter, the present invention will be described in detail with reference to the following examples. However, it should be understood that the present invention is not limited to the following examples.

Manufacturing example  1-1. Dichloroacetic acid ( Dichloroacetic  acid) and Ascorbic acid  Manufacture of calcium salt

129 mg of dichloroacetic acid was dissolved in 125 ml of distilled water to prepare a dichloroacetic acid solution and 176 mg of ascorbic acid was dissolved in 125 ml of distilled water to prepare an ascorbic acid solution. The ascorbic acid solution was added to the dichloroacetic acid solution with gentle stirring. Then, 105 mg of calcium carbonate (CaCO ₃ ) was added slowly and stirred at room temperature for 30 minutes, then gradually raised the reaction temperature to 60 ° C and allowed to react until no more CO ₂ was produced. Drying with a rotary evaporator and vacuum oven, removal of unreacted material with diethyl ether, and filtration, drying and pulverization were performed to obtain powdery dichloroacetic acid and ascorbic acid calcium salt. All reactions were carried out in the presence of nitrogen.

Manufacturing example  1-2. Ascorbic acid and Lactate  Manufacture of calcium salt

90 mg of L-lactic acid was dissolved in 125 ml of distilled water to prepare a lactic acid solution, and 176 mg of ascorbic acid was dissolved in 125 ml of distilled water to prepare an ascorbic acid solution. The ascorbic acid solution was slowly added to the lactic acid solution with stirring. Then, 105 mg of calcium carbonate (CaCO ₃ ) was added slowly and stirred at room temperature for 30 minutes, then gradually raised the reaction temperature to 60 ° C and allowed to react until no more CO ₂ was produced. Dried and pulverized by removing the unreacted material with a rotary evaporator and a vacuum oven and drying with diethyl ether, and obtaining a calcium salt of ascorbic acid and lactate in powder form. All reactions were carried out in the presence of nitrogen.

Manufacturing example  1-3. Dichloroacetic acid ( Dichloroacetic  acid) and Lactate  Manufacture of calcium salt

640 mg of dichloroacetic acid and 450 mg of L-lactic acid were dissolved in 10 ml of distilled water with stirring, 500 mg of calcium carbonate (CaCO ₃ ) was added slowly, and the mixture was stirred at room temperature for 30 minutes. Drying with a rotary evaporator and a vacuum oven, and removal of unreacted material with diethyl ether, followed by filtration, drying and pulverization, yielded a calcium salt of dichloroacetic acid and lactate in powder form.

Example  One

According to Preparation Example 1, the prepared calcium salt is combined with dichloroacetic acid and ascorbic acid dicalcium ion.

Example  2

The calcium salt according to Production Example 2, wherein the produced ascorbic acid and lactate are combined with calcium ions.

Example  3

According to Preparation Example 3, the prepared calcium salt in which the prepared dichloroacetic acid and lactate are combined with calcium ions.

Experimental Example  One. Calcium salt Cancer cell  The uptake effect and Cancer cell  pH change

The calcium salts of Examples 1 to 3 were each treated with cancer cells, and then the levels of intracellular calcium, lactate, ascorbic acid, and pH were analyzed to predict the influx levels of the respective calcium salts.

Experimental Example  1-1: Changes in calcium levels

A cancer cell culture medium supplemented with 5 x 10 ⁶ Number of human colon cancer cell lines (HCT-116) cell cultures by 37 ℃ and 5% CO ₂ conditions (10% FBS and 1% penicillin / streptomycin RPMI1640 medium containing) The calcium salts of Examples 1 to 3 were each treated with 1 mM and cultured for 24 hours. The cultured cancer cells were pulverized with a homogenizer and centrifuged. The concentration of calcium contained in the lysate was measured using a calcium assay kit (Biovision, San Francisco, Calif.). At this time, cancer cells not treated with calcium salt were used as a control group.

In order to observe changes in calcium level by fluorescence imaging, human colon cancer cell line (HCT-116) of 3 x 10 ⁴ cells was placed on a 6-well plate and cultured for 24 hours. Then, calcium of Examples 1 to 3 The salts were treated with 1 mM each, incubated for 4 hours, washed twice with DPBS, and incubated with Fluo 4-AM for 40 minutes. The calcium concentration in the cells was measured using a FACSCanto ^占 flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and a primary argon laser to evaluate intracellular calcium concentration. At this time, cancer cells not treated with calcium salt were used as a control group.

As shown in FIGS. 1A and 1B, the concentration of calcium in cancer cells treated with calcium salts of Examples 1 to 3 was increased. Thus, it was confirmed that the composition for treating cancer comprising an ionic compound bound to a metal ion according to the present invention can penetrate into cancer cells.

Experimental Example  1-2: Lactate  Level change

Cancer cell culture medium in (10% FBS and 1% penicillin / streptomycin RPMI1640 medium containing) 37 ℃ and 5% CO a 5 x cultured in _two conditions 10 ⁶ cells be of human colon cancer cell lines (HCT-116 and HT -29) were treated with 1 mM each of the calcium salts of Examples 1 to 3 and cultured for 24 hours. The cultured cells were pulverized with a homogenizer and centrifuged. The lactate concentration contained in the lysate was measured using a lactate assay kit (Biovision, San Francisco, Calif.) And is shown in FIG. At this time, as a control group, cancer cells which were not treated with anything were used.

As shown in FIG. 2, the concentrations of lactate in the cancer cells treated with the calcium salts of Examples 1 to 3 were increased. Accordingly, it has been confirmed that the composition for treating cancer, which comprises the ionic compound bound to the metal ion according to the present invention, can penetrate into cancer cells and increase the lactate concentration.

Experimental Example  1-3: Ascorbic acid  Level change

Cancer cell culture medium in (10% FBS and 1% penicillin / streptomycin RPMI1640 medium containing) 37 ℃ and 5% CO a 5 x cultured in _two conditions 10 ⁶ cells be of human colon cancer cell lines (HCT-116 and HT -29) were treated with 1 mM each of the calcium salts of Examples 1 and 2 and cultured for 24 hours. The cultured cancer cells were pulverized with a homogenizer and centrifuged. The concentration of ascorbic acid contained in the lysate was measured using an ascorbic acid assay kit (Biovision, San Francisco, Calif.) And shown in FIG. At this time, cancer cells that had not undergone any treatment were used as the control group. Cancer cells treated with ascorbic acid (1 mM) were used as Comparative Example 1, and cancer cells treated with calcium ascorbate (1 mM) Respectively.

As shown in FIG. 3, the concentration of ascorbic acid in the cancer cells treated in Examples 1 and 2 was increased, while the cancer cells treated in Comparative Examples 1 and 2 were the cancer cells treated in Examples 1 and 2 , The concentration of ascorbic acid increased. Thus, it was confirmed that the composition for treating cancer comprising an ionic compound bound to a metal ion according to the present invention is easy to penetrate into cancer cells.

Experimental Example  1-4: Cancer cell  Changes in pH

Cancer cell culture medium in (10% FBS and 1% penicillin / streptomycin RPMI1640 medium containing) 37 ℃ and 5% CO a 5 x cultured in _two conditions 10 ⁶ cells be of human colon cancer cell lines (HCT-116 and HT -29) were treated with calcium salts (1 mM) of Examples 1 to 3, respectively, and cultured for 24 hours. The cultured cell culture medium was measured using a pH detection kit (life technologies, CA) and shown in FIG. At this time, as a control group, cancer cells which were not treated with anything were used.

 As shown in FIG. 4, when the calcium salts of Examples 1 to 3 were treated, the intracellular pH was lowered to show acidity. In other words, it was found that the environment of cancer cells changed to acid due to the influx of calcium salt. This means that the calcium salt has become vulnerable to apoptosis.

Experimental Example  2: Cancer Intracellular  Effect of Calcium Salt on Metabolic Process

The calcium salts of Examples 1 to 3 were each treated with cancer cells to confirm the effect on cancer cell metabolism.

Experimental Example  2-1: Effect of calcium salt on the level of pyruvic acid

Cancer cell culture medium in (10% FBS and 1% penicillin / streptomycin RPMI1640 medium containing) 37 ℃ and 5% CO a 5 x cultured in _two conditions 10 ⁶ cells be of human colon cancer cell lines (HCT-116 and HT -29) were treated with calcium salts of Example 1 (1 mM), Example 2 (1 mM) and Example 3 (1 mM), respectively, and cultured for 24 hours. The cultured cells were pulverized with a homogenizer and centrifuged. The concentration of pyruvic acid contained in the lysate was measured using a pyruvic acid assay kit (Biovision, San Francisco, Calif.) And shown in FIG. At this time, as a control group, cancer cells which were not treated with anything were used.

As shown in FIG. 5, the concentrations of pyruvic acid in cancer cells treated with the calcium salts of Examples 1 to 3 were increased. Thus, it has been confirmed that the composition for treating cancer, which contains the ionic compound bound to the metal ion according to the present invention, can penetrate into cancer cells and increase the concentration of pyruvic acid.

Experimental Example  2-2: Alpha ketoglutamic acid (? ketoglutarate ; The effect of calcium salt on the level of α-KG

Cancer cell culture medium in (10% FBS and 1% penicillin / streptomycin RPMI1640 medium containing) 37 ℃ and 5% CO a 5 x cultured in _two conditions 10 ⁶ cells be of human colon cancer cell lines (HCT-116 and HT -29) were treated with calcium salts of Example 1 (1 mM), Example 2 (1 mM) and Example 3 (1 mM), respectively, and cultured for 24 hours. The cultured cells were pulverized with a homogenizer and centrifuged. The concentration of alpha-keto glutamic acid contained in the lysate was measured using an alpha-keto glutamic acid assay kit (Biovision, San Francisco, Calif.) And shown in FIG. At this time, as a control group, cancer cells which were not treated with anything were used.

As shown in FIG. 6, the concentrations of alpha-keto glutamic acid in the cancer cells treated with calcium salts of Examples 1 to 3 were increased. Accordingly, it has been confirmed that the composition for treating cancer, which comprises the ionic compound bound to the metal ion according to the present invention, penetrates into cancer cells and induces oxidative phosphorylation of mitochondria, thereby increasing the concentration of alpha-keto glutamic acid.

Experimental Example  2-3: PARP -1,? - catenin , VEGF (Vascular endothelial growth factor) and β-actin protein expression level

A cancer cell culture medium supplemented with 5 x 10 ⁶ Number of human colon cancer cell lines (HCT-116) cell cultures by 37 ℃ and 5% CO ₂ conditions (10% FBS and 1% penicillin / streptomycin RPMI1640 medium containing) Various concentrations of the calcium salts of Example 1 Example 2 and Example 3 were each treated and incubated for 24 hours. The cultured cells were pulverized with a homogenizer and centrifuged. The expression levels of poly (ADP-ribose) polymerase 1 (PARP-1), β -catenin, VEGF and β- The results are shown in Fig.

As shown in FIG. 7, the cancer cells treated with the calcium salts of Examples 1 to 3 had lower levels of PARP-1 expression. In general, PARP-1 is used as a cell death marker because cleavage occurs by caspase-3, which is activated when cells undergo apoptosis, which is a programmed cell death. The full length PAPR-1 was most greatly decreased at 0.18 mg / ml in Example 1, 0.34 mg / ml in Example 2, and 0.3 mg / ml in Example 3, and thus the cancer cell death was induced in a concentration- Respectively. Accordingly, it has been confirmed that the composition for treating cancer comprising an ionic compound bound to a metal ion according to the present invention can reduce the expression of full-length PAPR-1, thereby inducing the death of cancer cells.

 In addition, it was confirmed that the cancer cells treated with the calcium salts of Examples 1 to 3 reduced protein levels of? -Catenin in a concentration-dependent manner. β-catenin is a transcription factor that is mutated or overexpressed in various carcinomas such as colorectal, lung, breast, and ovarian cancers and plays an important role in cell growth, cancer metastasis, and survival, such as c-myc, cyclin D1, MMP7 and survivin It is known to regulate the expression of proteins. Accordingly, it has been confirmed that the composition for treating cancer comprising an ionic compound bound to a metal ion according to the present invention can inhibit the growth of cancer cells by decreasing the protein of? -Catenin.

In addition, it was confirmed that the cancer cells treated with the calcium salts of Examples 1 to 3 reduced the expression of VEGF in a concentration-dependent manner. On the other hand, VEGF signaling system plays an important role in cell growth, invasion and metastasis by regulating the lower MAPK signaling system and PI3K / Akt signaling system. In particular, it increases gene expression of matrix metalloproteinases (MMPs) essential for cancer cell metastasis and promotes cancer cell metastasis. Therefore, it has been confirmed that the composition for treating cancer comprising an ionic compound bound to a metal ion according to the present invention inhibits the action of a factor inducing angiogenesis and inhibits metastasis of cancer cells.

Experimental Example  2- 4: active oxygen  Change in expression level

Dichlorofluorescin diacetate (DCF-DA; Sigma, USA) was used as a fluorescent probe to measure changes in the concentration of reactive oxygen species in cancer cells treated with ionic compounds bound to metal ions according to the present invention. DCF-DA is oxidized by ROS in the presence of peroxides associated with intracellular hydrogen peroxide and converted to fluorescent DCF, resulting in green fluorescence. Therefore, the measurement of ROS was confirmed by DCF-DA. First, a cancer cell culture medium 5 x 10 ⁶ cells be of human colon cancer cell lines (HCT-116) in 37 ℃ and 5% CO ₂ conditions (10% FBS and 1% penicillin / streptomycin RPMI1640 medium containing) And cultured for 24 hours. After incubation, the cells were washed once with DPBS and 10 μM DCF-DA was incubated at 37 ° C for 30 minutes. DPBS, calcium salts of Examples 1, 2 and 3 were treated for 6 hours at various concentrations, and the intracellular ROS fluorescence was measured and analyzed and shown in FIG.

As shown in FIG. 8, cancerous cells treated with the calcium salts of Examples 1 to 3 showed more active oxygen indicating the possibility of inducing apoptosis than the control group treated with nothing.

Experimental Example  2- 5: Cell  Change of death level

To measure the concentration of active oxygen in the cancer cells to which the ionic compound bound to the metal ion according to the present invention was administered, a human colon cancer cell line (HCT-116) of 3 x 10 ⁴ cells was placed on a 6-well plate, After incubation, the calcium salts of the concentrations of Example 1 (1 mM), Example 2 (1 mM) and Example 3 (1 mM) were treated for 24 hours, washed twice with DPBS, and incubated with trypsin- separation and analysis are shown in Figure 9, by using the Annexin-V / PI stained with protocol ^{FACSCanto TM ⅱ flow cytometer (Becton-} Dickinson, Franklin Lakes, Nj, USA), primary argon laser measuring cell death as a.

In normally live cells, phosphatidyl serine (PS) is located inside the cell membrane. However, at the time of apoptosis, PS is exposed to the outside of the cell membrane, and annexin V binds with high affinity to PS to fluoresce. Propidium iodide (PI) enters the cell and stains the nucleus. Early apoptosis-progressing cells are stained only with annexin-V and not with PI, whereas late-stage apoptosis-progressing cells or necrosis- The integrity of the cell membrane is impaired and annexin-V and PI are stained simultaneously, and living cells are not stained in any of them. As shown in FIG. 9, cancer cells treated with the calcium salts of Examples 1 to 3 were more likely to induce apoptosis than the control group without any treatment.

Experimental Example  3: Evaluation of the effect on proliferative capacity of cancer cell line

The inhibitory effect on the viability of colorectal cancer, breast cancer and brain cancer cell line according to the calcium salt treatment of Examples 1 to 3 was examined.

Experimental Example  3-1: Proliferative capacity of cancer cell line ( MTT  assay

(DLD-1), a breast cancer cell line (MDA-MB-231), and a brain cancer cell line (U87MG) were each subdivided into 5 x 10 ⁶ cells in each well of a 96- The salt was added to each well with concentration (20 / / ml, 4 / / ㎖, 0.8 / / ㎖, 0.16 / / ㎖, 0.032 / / ㎖, 0.0064 / / ㎖, 0.00128 / / ㎖, 0.000256 / / And for comparison, ascorbic acid and dichloroacetic acid were also diluted in the same manner and added to the wells. The cells were cultured in a 5% CO ₂ incubator at 37 ° C for 72 hours. 50 μl of a 2 mg / ml MTT reagent was added thereto, followed by incubation at 37 ° C for 4 hours. The supernatant was removed using a centrifuge, 200 μl of DMSO was added to each well to dissolve the MTT staining precipitate, and the OD ₅₄₀ value was measured at 540 nm wavelength using an ELISA reader. The 50% inhibitory concentration (IC ₅₀ ) was defined as the concentration of the drug that allowed the survival rate to be 50%, and the IC ₅₀ value was shown in Table 1 below as an indicator of the anti-cancer effect.

division Colon cancer IC of cell line ₅₀
(Mg / ml) Breast cancer IC of cell line ₅₀
(Mg / ml) IC of brain cancer cell line ₅₀
(Mg / ml) Example 1 0.07 0.04 0.5 Example 2 0.07 0.05 0.6 Example 3 0.86 0.05 0.8 Dichloroacetic acid 1.21 0.43 3.71 Ascorbic acid 0.09 0.06 0.9

As shown in Table 1, the calcium salts of Examples 1 to 3 exhibited lower values of IC ₅₀ values for colorectal cancer, breast cancer and brain cancer cell lines than those of ascorbic acid and dichloroacetic acid, .

Experimental Example  3-2: Cancer cell line On community formation  Effect evaluation

(0 mM, 0.2 mM, 0.5 mM), dichloroacetic acid (0 mM, 0.2 mM, 0.5 mM) of human colorectal cancer cell line (HCT-116) and each ascorbic acid 2 mM, 5 mM) and the calcium salt of Example 3 (0 mM, 2 mM, 5 mM), and cultured for 72 hours. After completion of the culture, the cells were fixed, The cells were stained with mathoxylin, and the cancer cells in which the colonies were formed were observed and shown in Fig.

As shown in FIG. 10, all of the colorectal cancer cell lines without treatment of the calcium salt of Examples 1 to 3 formed several hundreds of clusters, but the calcium salts of Examples 1 to 3 were increased in the number of clusters And the calcium salts of Examples 1 to 3 were superior to those of ascorbic acid and dichloroacetic acid in inhibiting the colonization ability of colon cancer. As a result, the calcium salts of Examples 1 to 3 showed the effect of inhibiting colonization of colon cancer.

 Therefore, according to the results of Experimental Example 3, it can be seen that the composition for treating cancer, which includes the metal ion-bonded ionic compound according to the present invention, can reduce the survival rate of cancer cells such as colon cancer, breast cancer, I could.

Experimental Example  4: Known anticancer drugs and Example  Co-treatment of 1 to 3 calcium salts

The known anticancer agent and the calcium salt of Examples 1 to 3 were used in combination to examine the therapeutic effect on various cancer cell lines.

Experimental Example  4- 1: 5 -FU (5- Fluorouracil )Wow Example  Combined treatment effect

1 × 10 ³ cells were placed in 6-well plates containing RPMI 1640 medium, and the medium was replaced with the calcium salt of Example 1 (0.2 mM) (0.2 mM), the calcium salt of Example 3 (2 mM), and 5 [mu] M of 5-FU were individually treated in each well and a 5 [mu] M concentration of 5-FU and the calcium salt of Example 1 0.2 mM), 5-FU at a concentration of 5 μM and calcium salt (0.2 mM) of Example 2, 5-FU at a concentration of 5 μM and calcium salt of Example 3 (2 mM) The formation ability was compared and shown in Fig. As a control group, a human colorectal cancer cell line (HCT-116) was used which was not treated with any drug.

As shown in FIG. 11, the colon cancer cell line which had not undergone any treatment formed a hundred clusters, but the combination of 5-FU and the calcium salts of Examples 1 to 3 was more effective than 5-FU alone It was observed that the effect of inhibiting the colonization of cancer was more excellent

Experimental Example  5: Through animal models Example  For calcium salts Anticancer effect  Verification

Experimental Example  5-1: Animal model ( orthotropic  model) carcinoma  Formative animal model construction

 A549 / LUC cells and DLD-1 cells were subcultured to construct orthotopic xenografts and orthotopic xenografts, and then the above cancer cells were injected into the lungs and large intestine of the mice, respectively Respectively.

Since the cancer cells were injected directly into the mouse organs, the growth of the cancer was not confirmed by mouse appearance. Therefore, in the case of animal models injected with A549 / LUC cells, D-Luciferin was injected intraperitoneally The growth saturation of cancer was confirmed by luminescence imaging measurement using IVIS spectral imaging system (Xenogen). On the other hand, in case of animal model injected with DLD-1 cells, . In the case of the animal model injected with A549 / LUC cells, about 4 weeks later, when the intensity of luminescence was found to be about 10 ⁷ photons / s / cm ² / sr, the calcium salt of Examples 1 to 3 was administered, In the animal model injected with DLD-1 cells, the anticancer effect was observed after about 7 weeks, when the calcium salt of Example 1 was administered at the stage where the expression of the colon cancer was at the end stage.

That is, a DLD-1 orthotopic model was constructed by transplanting DLD-1 cells into the large intestine, and a lung cancer mouse model (A549 / LUC orthotopic model) was constructed by transplanting a heterozygote in the lung. Then, the drug was administered to each mouse model as shown in Table 2 below.

Animal experiment model drug density Drug administration method Population Mouse model (DLD-1 orthotopic model) Control group - Once daily (Qdx4), intravenously (IV), 1 week 4 Mouse model (DLD-1 orthotopic model) Example 1 100mg / kg (Dissolved in saline) Once daily (Qdx4), intravenously (IV), 1 week 4 Mouse model (A549 / LUC orthotopic model) Control group - Once daily (Qdx4), intravenously (IV), 5 weeks 3 Mouse model (A549 / LUC orthotopic model) Example 1 100mg / kg (Dissolved in saline) Once daily (Qdx4), intravenously (IV), 5 weeks 3 Mouse model (A549 / LUC orthotopic model) Example 2 100mg / kg (Dissolved in saline) Once daily (Qdx4), intravenously (IV), 5 weeks 3 Mouse model (A549 / LUC orthotopic model) Example 3 100mg / kg (Dissolved in saline) Once daily (Qdx4), intravenously (IV), 5 weeks 3

Experimental Example  5-2: In an animal model injected with calcium salt Anticancer effect  And Cancer  Change (1)

The calcium salt of Example 1 was administered to the same mouse model (DLD-1 orthotopic model) constructed in Experimental Example 5-1, and after 1 week, the calcium salt was observed to observe the growth state of cancer cells, The cancerous tissue weight was measured to confirm the anticancer efficacy of the substance of the present invention in vivo and shown in FIG. 12B.

As shown in FIGS. 12A and 12B, in all the mouse models (DLD-1 orthotopic model) not treated with the calcium salt of Example 1, the calcium salt of Example 1 was observed in all mouse models (DLD -1 orthotopic model), the growth of cancer cells was significantly inhibited.

Experimental Example  5-3: In an animal model injected with calcium salt Anticancer effect  And Cancer  Change (2)

The calcium salts of Examples 1 to 3 were each administered to the same mouse model (A549 / LUC orthotopic model) constructed in Experimental Example 5-1, and the tissue distribution, metastasis and anticancer efficacy of the inventive substances were confirmed in vivo An image photograph is shown in Fig. In order to more accurately quantify each imaging result, the in vivo image was measured by observing the region of interest (ROI) of the IVIS spectrum (Xenogen), and FIG. 14 shows the results. FIG. 15 shows the mouse model (A549 / LUC orthotopic model).

13, 14 and 15, it was confirmed that the calcium salts of Examples 1 to 3 have excellent anticancer efficacy, transit inhibition ability and excellent survival rate as compared with the control group. In particular, Example 1 did not show any growth and metastasis of cancer tissues at all, not only during the drug administration period but also after stopping the drug administration, which suggests that the mitochondria in the cancer cells have been reformed.

Therefore, it has been confirmed from the above-mentioned experiment results that the ionic compound bound to the metal ion according to the present invention can increase the uptake to cancer cells and can lower the pH in the cancer cell to acidify it. It was confirmed that the ionic compound in which two kinds of compounds are bonded to a metal ion rather than each of the compounds (ascorbic acid or dichloaroacetic acid) was more effective in killing cancer cells.

In addition, it has been confirmed that the ionic compound can inhibit the corresponding action of cancer cells by increasing pyruvic acid and alpha-keto glutamic acid. Through the changes in the amount of β-catenin, PARP, and VEGF expression, cancer cell proliferation and metastasis Of the total population. In addition, it has been confirmed through experiments of proliferation ability of cancer cell line that it can exhibit more excellent anticancer effect when it is co-administered with conventional anticancer drug.

Claims (14)

A pharmaceutical composition for treating cancer, which comprises, as an active ingredient, an ionic compound in which two compounds selected from ascorbic acid, dichloroacetic acid and lactate are combined with metal ions of Ca ²⁺ . delete The method according to claim 1,
Wherein the pharmaceutical composition is used for radiation therapy or in combination therapy with an anticancer agent. The method of claim 3,
Wherein the radiation is irradiated to a cancer patient at an irradiation dose of 2 to 10 Gy per day, and is treated in combination with the pharmaceutical composition. The method of claim 3,
The anticancer agent may be selected from the group consisting of imatinib, 5-fluorouracil, irinotecan, sunitinib, oxaliplatin, paclitaxel, lapatinib, Trastuzumab, Herceptin, Gefitinib, Erlotinib, Methotrexate, Carboplatin, Docetaxel, Everolimus, Sorafenib, Carbonic anhydride, Inhibitors of carbonic anhydrase, inhibitors of monocarboxylate transporters, Pembrolizumab, Atezolizumab, PD-1 family anticancer agents, Nivolumab, , Inhibitors of PARP-1 (poly (ADP-ribose) polymerase 1), inhibitors of PARP-2 (poly (ADP-ribose) polymerase 2), Olaparib, Rucaparib, Niraparib, , Bevacizumab, and a VEGF inhibitor < RTI ID = 0.0 > Will a pharmaceutical composition for the treatment of cancer and more cancer. The method according to claim 1,
Wherein the cancer is a cancer selected from the group consisting of lung cancer, breast cancer, colon cancer, stomach cancer, brain cancer, pancreatic cancer, thyroid cancer, skin cancer, bone cancer, lymphoma, uterine cancer, cervical cancer, renal cancer and melanoma. The method according to claim 1,
Wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or diluent. The method according to claim 1,
Wherein the pharmaceutical composition is formulated in the form of a solution, a powder, an aerosol, an injection, a solution (ring gel), a patch, a capsule, a pill, a tablet, a depot or a suppository. A pharmaceutical composition for inhibiting cancer metastasis which comprises, as an active ingredient, an ionic compound in which two kinds of compounds selected from ascorbic acid, dichloroacetic acid and lactate are combined with a metal ion of Ca ²⁺ . delete 10. The method of claim 9,
Wherein the cancer is a cancer selected from the group consisting of lung cancer, breast cancer, colon cancer, stomach cancer, brain cancer, pancreatic cancer, thyroid cancer, skin cancer, bone cancer, lymphoma, uterine cancer, cervical cancer, kidney cancer and melanoma. Prevention or amelioration of cancer-related fatigue comprising, as an active ingredient, an ionic compound in which two compounds selected from ascorbic acid, dichloroacetic acid and lactate are combined with metal ions of Ca ²⁺ ≪ / RTI > A food composition for improving cancer, which comprises, as an active ingredient, an ionic compound in which two compounds selected from ascorbic acid, dichloroacetic acid and lactate are combined with a metal ion of Ca ²⁺ . 14. The method of claim 13,
Wherein the food composition is manufactured from a health functional food in the form of a snack, a drink, a liquor, a fermented food, a canned food, a milk processed food, a meat processed food or a noodle processed food.

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